Compounds and methods of making sterols using diols

ABSTRACT

Compounds and methods of synthesizing oxysterols are provided. The compounds and methods provided allow the oxysterol to be safely produced at a high yield. The compounds and methods provided can produce the oxysterol in a stereoselective manner.

This application claims the benefit of the filing date of U.S.Provisional Application No. 62/089,607, filed Dec. 9, 2014, entitled“COMPOUNDS AND METHODS OF MAKING STEROLS USING DIOLS”; and U.S.Provisional Application No. 62/089,600, filed Dec. 9, 2014, entitled“COMPOUNDS AND METHODS INVOLVING STEROLS”. These entire disclosures arehereby incorporated by reference into the present disclosure.

BACKGROUND

Biologics are commonly employed to promote bone growth in medicalapplications including fracture healing and surgical management ofspinal disorders. Spine fusion is often performed by orthopedic surgeonsand neurosurgeons alike to address degenerative disc disease andarthritis affecting the lumbar and cervical spine. Historically,autogenous bone grafting, commonly taken from the iliac crest of thepatient, has been used to augment fusion between vertebral levels.

One protein that is osteogenic and commonly used to promote spine fusionis recombinant human bone morphogenetic protein-2 (rhBMP-2). Its use hasbeen approved by the US Food and Drug Administration (FDA) forsingle-level anterior lumbar interbody fusion. The use of rhBMP-2 hasincreased significantly since this time and indications for its use haveexpanded to include posterior lumbar spinal fusion as well as cervicalspine fusion.

Oxysterols form a large family of oxygenated derivatives of cholesterolthat are present in the circulation, and in human and animal tissues.Oxysterols have been found to be present in atherosclerotic lesions andplay a role in various physiologic processes, such as cellulardifferentiation, inflammation, apoptosis, and steroid production. Somenaturally occurring oxysterols have robust osteogenic properties and canbe used to grow bone. The most potent osteogenic naturally occurringoxysterol, 20(S)-hydroxycholesterol, is both osteogenic andanti-adipogenic when applied to multipotent mesenchymal cells capable ofdifferentiating into osteoblasts and adipocytes.

One such oxysterol is Oxy133 or (3S,5S,6S,8R,9S,10R,13S,14S,17S)17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol,which exhibits the following structures:

To synthesize Oxy133, often there are complex, multi-step chemicalreactions that are difficult to carry out in a single container. Forexample, to synthesize Oxy133 there may be utilization of variousprotection reagents to protect end groups as the molecule is beingsynthesized. In addition, various deprotection reagents are alsoutilized that increase cost, reduce safety and have an adverseenvironmental impact. Further, the route of synthesis of OXY133 can havevery low yield less than 30%.

Therefore, there is a need for a cost effective method of synthesizingOxy133 for use in promoting osteogenesis, osteoinduction and/orosteoconduction. Methods of synthesizing Oxy133 having a high yield andimproved process safety would be beneficial. Methods for synthesizingOxy133 from endogenous starting material, which is stereoselective,would also be beneficial.

SUMMARY

Compounds and methods of synthesizing Oxy133 are provided for use inpromoting osteogenesis, osteoinduction and/or osteoconduction. Methodsof synthesizing Oxy133 having high yields and improved process safetyare also provided. Methods for synthesizing Oxy133 that arestereoselective are also provided. Methods of synthesizing Oxy133 thathave reduced environmental impact and have low product cost are alsoprovided.

In some embodiments, there is a compound comprising the structure:

or a pharmaceutically acceptable salt, hydrate or solvate thereof,wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom.

In some embodiments there is a method of making a sterol, the methodcomprising reacting an organometallic compound with pregnenolone orpregnenolone acetate to form the sterol, the sterol having the formula:

or a pharmaceutically acceptable salt, hydrate or solvate thereof,wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom.

In some embodiments, there is a method of making an oxysterol, themethod comprising reacting a diol having the formula:

with borane and hydrogen peroxide to form the oxysterol or apharmaceutically acceptable salt, hydrate or solvate thereof having theformula:

wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom, and wherein R2 comprises an aliphatic or cyclicsubstituent having at least one carbon atom.

In some embodiments, there is a method of making an oxysterol, themethod comprising reacting a diol having the formula:

with a borane compound to form the oxysterol or a pharmaceuticallyacceptable salt, hydrate or solvate thereof having the formula:

wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom, and R2 comprises an aliphatic or cyclic substituenthaving at least one carbon atom.

In some embodiments, there is a method of making an oxysterol, themethod comprising reacting a diol having the formula:

with borane, hydrogen peroxide and tetrahydrofuran to form the oxysterolor a pharmaceutically acceptable salt, hydrate or solvate thereof havingthe formula:

wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom, and R2 comprises an aliphatic or cyclic substituenthaving at least one carbon atom.

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawings where:

FIG. 1 illustrates a step-wise reaction for synthesizing Oxy133 withstarting reactants comprising pregnenolone acetate, as shown in oneembodiment of this disclosure. The pregnenolone is reacted with anorganometallic compound to produce a sterol or diol having two hydroxylgroups. The sterol or diol is then reacted with borane and hydrogenperoxide and purified to produce Oxy133;

FIG. 2 is a graphic illustration of the ¹H NMR data obtained fromisolated and purified Oxy133;

FIG. 3 is a graphic illustration of the ¹³C NMR data obtained fromOxy133;

FIG. 4 is a graphic illustration of the infrared spectroscopy dataobtained from Oxy133;

FIG. 5 is a graphic illustration of the mass spectroscopy data obtainedfrom Oxy133;

FIG. 6 is a graphic illustration of ¹H NMR data obtained from theintermediary sterol or diol to synthesize Oxy133;

FIG. 7 is a graphic illustration of ¹³C NMR data obtained from theintermediary sterol or diol to synthesize Oxy133.

It is to be understood that the figures are not drawn to scale. Further,the relation between objects in a figure may not be to scale, and may infact have a reverse relationship as to size. The figures are intended tobring understanding and clarity to the structure of each object shown,and thus, some features may be exaggerated in order to illustrate aspecific feature of a structure.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present application. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the present application are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Moreover, all ranges disclosedherein are to be understood to encompass any and all sub ranges subsumedtherein. For example, a range of “1 to 10” includes any and all subranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all sub ranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

DEFINITIONS

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “an alkanolamine” includes one, two, three or morealkanolamines.

The term “bioactive agent” as used herein is generally meant to refer toany substance that alters the physiology of a patient. The term“bioactive agent” may be used interchangeably herein with the terms“therapeutic agent,” “therapeutically effective amount,” and “activepharmaceutical ingredient”, “API” or “drug”.

The term “biodegradable” includes compounds or components that willdegrade over time by the action of enzymes, by hydrolytic action and/orby other similar mechanisms in the human body. In various embodiments,“biodegradable” includes that components can break down or degradewithin the body to non-toxic components as cells (e.g., bone cells)infiltrate the components and allow repair of the defect. By“bioerodible” it is meant that the compounds or components will erode ordegrade over time due, at least in part, to contact with substancesfound in the surrounding tissue, fluids or by cellular action. By“bioabsorbable” it is meant that the compounds or components will bebroken down and absorbed within the human body, for example, by a cellor tissue. “Biocompatible” means that the compounds or components willnot cause substantial tissue irritation or necrosis at the target tissuesite and/or will not be carcinogenic.

The term “alkyl” as used herein, refers to a saturated or unsaturated,branched, straight-chain or cyclic monovalent hydrocarbon radicalderived by the removal of one hydrogen atom from a single carbon atom ofa parent alkane, alkene or alkyne. Typical alkyl groups include, but arenot limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl;propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls suchas butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkenyl”and/or “alkynyl” is used, as defined below. In some embodiments, thealkyl groups are (C1-C40) alkyl. In some embodiments, the alkyl groupsare (C1-C6) alkyl.

The term “alkanyl” as used herein refers to a saturated branched,straight-chain or cyclic alkyl radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane. Typicalalkanyl groups include, but are not limited to, methanyl; ethenyl;propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl,etc.; butyanyls such as butan-1-yl, butan-2-yl (sec-butyl),2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl),cyclobutan-1-yl, etc.; and the like. In some embodiments, the alkanylgroups are (C1-C40) alkanyl. In some embodiments, the alkanyl groups are(C1-C6) alkanyl.

The term “alkenyl” as used herein refers to an unsaturated branched,straight-chain or cyclic alkyl radical having at least one carbon-carbondouble bond derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkene. The radical may be in either the cis ortrans conformation about the double bond(s). Typical alkenyl groupsinclude, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In some embodiments, the alkenyl group is (C2-C40)alkenyl. In some embodiments, the alkenyl group is (C2-C6) alkenyl.

The term “alkynyl” as used herein refers to an unsaturated branched,straight-chain or cyclic alkyl radical having at least one carbon-carbontriple bond derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkyne. Typical alkynyl groups include, but arenot limited to, ethynyl; propynyls such as prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-3-yn-1-yl,etc.; and the like. In some embodiments, the alkynyl group is (C2-C40)alkynyl. In some embodiments, the alkynyl group is (C2-C6) alkynyl.

The term “alkyldiyl” as used herein refers to a saturated orunsaturated, branched, straight-chain or cyclic divalent hydrocarbonradical derived by the removal of one hydrogen atom from each of twodifferent carbon atoms of a parent alkane, alkene or alkyne, or by theremoval of two hydrogen atoms from a single carbon atom of a parentalkane, alkene or alkyne. The two monovalent radical centers or eachvalency of the divalent radical center can form bonds with the same ordifferent atoms. Typical alkyldiyls include, but are not limited tomethandiyl; ethyldiyls such as ethan-1,1-diyl, ethan-1,2-diyl,ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as propan-1,1-diyl,propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl, cyclopropan-1,1-diyl,cyclopropan-1,2-diyl, prop-1-en-1,1-diyl, prop-1-en-1,2-diyl,prop-2-en-1,2-diyl, prop-1-en-1,3-diyl cycloprop-1-en-1,2-diyl,cycloprop-2-en-1,2-diyl, cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl,etc.; butyldiyls such as, butan-1,1-diyl, butan-1,2-diyl,butan-1,3-diyl, butan-1,4-diyl, butan-2,2-diyl,2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl, cyclobutan-1,1-diyl;cyclobutan-1,2-diyl, cyclobutan-1,3-diyl, but-1-en-1,1-diyl,but-1-en-1,2-diyl, but-1-en-1,3-diyl, but-1-en-1,4-diyl,2-methyl-prop-1-en-1,1-diyl, 2-methanylidene-propan-1,1-diyl,buta-1,3-dien-1,1-diyl, buta-1,3-dien-1,3-diyl, cyclobut-1-en-1,2-diyl,cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Insome embodiments, the alkyldiyl group is (C1-C40) alkyldiyl. In someembodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also contemplatedare saturated acyclic alkanyldiyl radicals in which the radical centersare at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl(ethano); propan-1,3-diyl(propano);butan-1,4-diyl(butano); and the like (also referred to as alkylenos,defined infra).

The term “alkyleno” as used herein refers to a straight-chain alkyldiylradical having two terminal monovalent radical centers derived by theremoval of one hydrogen atom from each of the two terminal carbon atomsof straight-chain parent alkane, alkene or alkyne. Typical alkylenogroups include, but are not limited to, methano; ethylenos such asethano, etheno, ethyno; propylenos such as propano, prop[1]eno,propa[1,2]dieno, prop[1]yno, etc.; butylenos such as butano, but[1]eno,but[2]eno, buta[1,3]dieno, but[1]yno, but[2]yno, but[1,3]diyno, etc.;and the like. Where specific levels of saturation are intended, thenomenclature alkano, alkeno and/or alkyno is used. In some embodiments,the alkyleno group is (C1-C40) alkyleno. In some embodiments, thealkyleno group is (C1-C6) alkyleno.

The terms “heteroalkyl,” “heteroalkanyl,” “heteroalkenyl,”“heteroalkanyl,” “heteroalkyldiyl” and “heteroalkyleno” as used hereinrefer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkylenoradicals, respectively, in which one or more of the carbon atoms areeach independently replaced with the same or different heteroatomicgroups. Typical heteroatomic groups which can be included in theseradicals include, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—,—NR′, ═N—N═, —N═N—, —N(O)N—, —N═N—NR′—, —PH—, —P(O)2-, —O—P(O)2-, —SH2-,—S(O)2-, or the like, where each R′ is independently hydrogen, alkyl,alkanyl, alkenyl, alkynyl, aryl, arylaryl, arylalkyl, heteroaryl,heteroarylalkyl or heteroaryl-heteroaryl as defined herein.

The term “aryl” as used herein refers to a monovalent aromatichydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, radicals derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like. In some embodiments, the aryl group is (C5-C14) aryl or a(C5-C10) aryl. Some preferred aryls are phenyl and naphthyl.

The term “aryldiyl” as used herein refers to a divalent aromatichydrocarbon radical derived by the removal of one hydrogen atom fromeach of two different carbon atoms of a parent aromatic ring system orby the removal of two hydrogen atoms from a single carbon atom of aparent aromatic ring system. The two monovalent radical centers or eachvalency of the divalent center can form bonds with the same or differentatom(s). Typical aryldiyl groups include, but are not limited to,divalent radicals derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorine, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In someembodiments, the aryldiyl group is (C5-C14) aryldiyl or (C5-C10)aryldiyl. For example, some preferred aryldiyl groups are divalentradicals derived from benzene and naphthalene, especiallyphena-1,4-diyl, naphtha-2,6-diyl and naphtha-2,7-diyl.

The term “arydeno” as used herein refers to a divalent bridge radicalhaving two adjacent monovalent radical centers derived by the removal ofone hydrogen atom from each of two adjacent carbon atoms of a parentaromatic ring system. Attaching an aryleno bridge radical, e.g. benzeno,to a parent aromatic ring system, e.g. benzene, results in a fusedaromatic ring system, e.g. naphthalene. The bridge is assumed to havethe maximum number of non-cumulative double bonds consistent with itsattachment to the resultant fused ring system. In order to avoiddouble-counting carbon atoms, when an aryleno substituent is formed bytaking together two adjacent substituents on a structure that includesalternative substituents, the carbon atoms of the aryleno bridge replacethe bridging carbon atoms of the structure. As an example, consider thefollowing structure:

wherein R¹, when taken alone is hydrogen, or when taken together with R²is (C5-C14) aryleno; and R², when taken alone is hydrogen, or when takentogether with R¹ is (C5-C14) aryleno.

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is C6 aryleno (benzeno), the resultantcompound is naphthalene. When R¹ taken together with R² is C10 aryleno(naphthaleno), the resultant compound is anthracene or phenanthrene.Typical aryleno groups include, but are not limited to, aceanthryleno,acenaphthyleno, acephenanthtyleno, anthraceno, azuleno, benzeno (benzo),chryseno, coroneno, fluorantheno, fluoreno, hexaceno, hexapheno,hexyleno, as-indaceno, s-indaceno, indeno, naphthalene (naphtho),octaceno, octapheno, octaleno, ovaleno, penta-2,4-dieno, pentaceno,pentaleno, pentapheno, peryleno, phenaleno, phenanthreno, piceno,pleiadeno, pyreno, pyranthreno, rubiceno, triphenyleno, trinaphthaleno,and the like. Where a specific connectivity is intended, the involvedbridging carbon atoms (of the aryleno bridge) are denoted in brackets,e.g., [1,2]benzeno ([1,2]benzo), [1,2]naphthaleno, [2,3]naphthaleno,etc. Thus, in the above example, when R¹ taken together with R² is[2,3]naphthaleno, the resultant compound is anthracene. When R¹ takentogether with R² is [1,2]naphthaleno, the resultant compound isphenanthrene. In a preferred embodiment, the aryleno group is (C5-C14),with (C5-C10) being even more preferred.

The term “arylaryl” as used herein refers to a monovalent hydrocarbonradical derived by the removal of one hydrogen atom from a single carbonatom of a ring system in which two or more identical or non-identicalparent aromatic ring systems are joined directly together by a singlebond, where the number of such direct ring junctions is one less thanthe number of parent aromatic ring systems involved. Typical arylarylgroups include, but are not limited to, biphenyl, triphenyl,phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. When thenumber of carbon atoms comprising an arylaryl group is specified, thenumbers refer to the carbon atoms comprising each parent aromatic ring.For example, (C1-C14) arylaryl is an arylaryl group in which eacharomatic ring comprises from 5 to 14 carbons, e.g., biphenyl, triphenyl,binaphthyl, phenylnaphthyl, etc. In some instances, each parent aromaticring system of an arylaryl group is independently a (C5-C14) aromatic ora (C1-C10) aromatic. Some preferred are arylaryl groups in which all ofthe parent aromatic ring systems are identical, e.g., biphenyl,triphenyl, binaphthyl, trinaphthyl, etc.

The term “biaryl” as used herein refers to an arylaryl radical havingtwo identical parent aromatic systems joined directly together by asingle bond. Typical biaryl groups include, but are not limited to,biphenyl, binaphthyl, bianthracyl, and the like. In some instances, thearomatic ring systems are (C5-C14) aromatic rings or (C5-C10) aromaticrings. One preferred biaryl group is biphenyl.

The term “arylalkyl” as used herein refers to an acyclic alkyl radicalin which one of the hydrogen atoms bonded to a carbon atom, typically aterminal or spa carbon atom, is replaced with an aryl radical. Typicalarylalkyl groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In some embodiments, the arylalkyl group is(C6-C40) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C26) and the aryl moiety is (C5-C14). In somepreferred embodiments the arylalkyl group is (C6-C13), e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) andthe aryl moiety is (C5-C10).

The term “heteroaryl” as used herein refers to a monovalentheteroaromatic radical derived by the removal of one hydrogen atom froma single atom of a parent heteroaromatic ring system. Typical heteroarylgroups include, but are not limited to, radicals derived from acridine,arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan,imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. In some embodiments, the heteroaryl group is a 5-14 memberedheteroaryl, with 5-10 membered heteroaryl being particularly preferred.Some preferred heteroaryl radicals are those derived from parentheteroaromatic ring systems in which any ring heteroatoms are nitrogens,such as imidazole, indole, indazole, isoindole, naphthyridine,pteridine, isoquinoline, phthalazine, purine, pyrazole, pyrazine,pyridazine, pyridine, pyrrole, quinazoline, quinoline, etc.

The term “heteroaryldiyl” refers to a divalent heteroaromatic radicalderived by the removal of one hydrogen atom from each of two differentatoms of a parent heteroaromatic ring system or by the removal of twohydrogen atoms from a single atom of a parent heteroaromatic ringsystem. The two monovalent radical centers or each valency of the singledivalent center can form bonds with the same or different atom(s).Typical heteroaryldiyl groups include, but are not limited to, divalentradicals derived from acridine, arsindole, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In some embodiments, theheteroaryldiyl group is 5-14 membered heteroaryldiyl or a 5-10 memberedheteroaryldiyl. Some preferred heteroaryldiyl groups are divalentradicals derived from parent heteroaromatic ring systems in which anyring heteroatoms are nitrogens, such as imidazole, indole, indazole,isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine,pyrazole, pyrazine, pyridazine, pyridine, pyrrole, quinazoline,quinoline, etc.

The term “heteroaryleno” as used herein refers to a divalent bridgeradical having two adjacent monovalent radical centers derived by theremoval of one hydrogen atom from each of two adjacent atoms of a parentheteroaromatic ring system. Attaching a heteroaryleno bridge radical,e.g. pyridino, to a parent aromatic ring system, e.g. benzene, resultsin a fused heteroaromatic ring system, e.g., quinoline. The bridge isassumed to have the maximum number of non-cumulative double bondsconsistent with its attachment to the resultant fused ring system. Inorder to avoid double-counting ring atoms, when a heteroarylenosubstituent is formed by taking together two adjacent substituents on astructure that includes alternative substituents, the ring atoms of theheteroaryleno bridge replace the bridging ring atoms of the structure.As an example, consider the following structure:

wherein R¹, when taken alone is hydrogen, or when taken together with R²is 5-14 membered heteroaryleno; and R², when taken alone is hydrogen, orwhen taken together with R¹ is 5-14 membered heteroaryleno;

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R1 taken together with R² is a 6-membered heteroaryleno pyridino),the resultant compound is isoquinoline, quinoline or quinolizine. WhenR¹ taken together with R² is a 10-membered heteroaryleno (e.g.,isoquinoline), the resultant compound is, e.g., acridine orphenanthridine. Typical heteroaryleno groups include, but are notlimited to, acridino, carbazolo, β-carbolino, chromeno, cinnolino,furan, imidazolo, indazoleno, indoleno, indolizino, isobenzofurano,isochromeno, isoindoleno, isoquinolino, isothiazoleno, isoxazoleno,naphthyridino, oxadiazoleno, oxazoleno, perimidino, phenanthridino,phenanthrolino, phenazino, phthalazino, pteridino, purino, pyrano,pyrazino, pyrazoleno, pyridazino, pyridino, pyrimidino, pyrroleno,pyrrolizino, quinazolino, quinolino, quinolizino, quinoxalino,tetrazoleno, thiadiazoleno, thiazoleno, thiopheno, triazoleno, xantheno,or the like. Where a specific connectivity is intended, the involvedbridging atoms (of the heteroaryleno bridge) are denoted in brackets,e.g., [1,2]pyridino, [2,3]pyridino, [3,4]pyridino, etc. Thus, in theabove example, when R¹ taken together with R² is [1,2]pyridino, theresultant compound is quinolizine. When R¹ taken together with R2 is[2,3]pyridino, the resultant compound is quinoline. When R¹ takentogether with R² is [3,4]pyridino, the resultant compound isisoquinoline. In preferred embodiments, the heteroaryleno group is 5-14membered heteroaryleno or 5-10 membered heteroaryleno. Some preferredheteroaryleno radicals are those derived from parent heteroaromatic ringsystems in which any ring heteroatoms are nitrogens, such as imidazolo,indolo, indazolo, isoindolo, naphthyridino, pteridino, isoquinolino,phthalazino, purino, pyrazolo, pyrazino, pyridazino, pyndmo, pyrrolo,quinazolino, quinolino, etc.

The term “heteroaryl-heteroaryl” as used herein refers to a monovalentheteroaromatic radical derived by the removal of one hydrogen atom froma single atom of a ring system in which two or more identical ornon-identical parent heteroaromatic ring systems are joined directlytogether by a single bond, where the number of such direct ringjunctions is one less than the number of parent heteroaromatic ringsystems involved. Typical heteroaryl-heteroaryl groups include, but arenot limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc.When the number of ring atoms are specified, the numbers refer to thenumber of atoms comprising each parent heteroaromatic ring systems. Forexample, 5-14 membered heteroaryl-heteroaryl is a heteroaryl-heteroarylgroup in which each parent heteroaromatic ring system comprises from 5to 14 atoms, e.g., bipyridyl, tripyridyl, etc. In some embodiments, eachparent heteroaromatic ring system is independently a 5-14 memberedheteroaromatic, more preferably a 5-10 membered heteroaromatic. Alsopreferred are heteroaryl-heteroaryl groups in which all of the parentheteroaromatic ring systems are identical. Some preferredheteroaryl-heteroaryl radicals are those in which each heteroaryl groupis derived from parent heteroaromatic ring systems in which any ringheteroatoms are nitrogens, such as imidazole, indole, indazole,isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine,pyrazole, pyrazine, pyridazine, pyridine, pyrrole, quinazoline,quinoline, etc.

The term “biheteroaryl” as used herein refers to a heteroaryl-heteroarylradical having two identical parent heteroaromatic ring systems joineddirectly together by a single bond. Typical biheteroaryl groups include,but are not limited to, bipyridyl, bipurinyl, biquinolinyl, and thelike. In some embodiments, the heteroaromatic ring systems are 5-14membered heteroaromatic rings or 5-10 membered heteroaromatic rings.Some preferred biheteroaryl radicals are those in which the heteroarylgroups are derived from a parent heteroaromatic ring system in which anyring heteroatoms are nitrogens, such as biimidazolyl, biindolyl,biindazolyl, biisoindolyl, binaphthyridinyl, bipteridinyl,biisoquinolinyl, biphthalazinyl, bipurinyl, bipyrazolyl, bipyrazinyl,bipyridazinyl, bipyridinyl, bipyrrolyl, biquinazolinyl, biquinolinyl,etc.

The term “heteroarylalkyl” as used herein refers to an acyclic alkylradical in which one of the hydrogen atoms bonded to a carbon atom,typically a terminal or sp2 carbon atom, is replaced with a heteroarylradical. Where specific alkyl moieties are intended, the nomenclatureheteroarylalkanyl, heteroarylakenyl and/or heterorylalkynyl is used. Insome embodiments, the heteroarylalkyl group is a 6-20 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of theheteroarylalkyl is 1-6 membered and the heteroaryl moiety is a5-14-membered heteroaryl. In some preferred embodiments, theheteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a5-10 membered heteroaryl.

The term “substituted” as used herein refers to a radical in which oneor more hydrogen atoms are each independently replaced with the same ordifferent substituent(s). Typical substituents include, but are notlimited to, —X, —R, —O—, ═O, —OR, —O—OR, —SR, —S—, ═S, —NRR, ═NR,perhalo (C1-C6) alkyl, —CX3, —CF3, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO2, ═N2, —N3, —S(O)2O—, —S(O)2OH, —S(O)2R, —C(O)R, —C(O)X, —C(S)R,—C(S)X, —C(O)OR, —C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRRand —C(NR)NRR, where each X is independently a halogen (e.g., —F or —Cl)and each R is independently hydrogen, alkyl, alkanyl, alkenyl, alkanyl,aryl, arylalkyl, arylaryl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl, as defined herein. The actual substituentsubstituting any particular group will depend upon the identity of thegroup being substituted.

The term “solvate” as used herein refers to an aggregate or complex thatcomprises one or more molecules of a compound of the disclosure with oneor more molecules of solvent. Examples of solvents that form solvatesinclude, but are not limited to, water, isopropanol, ethanol, methanol,DMSO, ethyl acetate, acetic acid, and ethanolamine. The term “hydrate”refers to the aggregate or complex where the solvent molecule is water.The solvent may be inorganic solvents such as for example water in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent, such as ethanol. Thus, the compounds of the presentdisclosure may exist as a hydrate, including a monohydrate, dihydrate,hemihydrate, sesquihydrate, trihydrate, tetrahydrate or the like, aswell as the corresponding solvated forms. The compound of the disclosuremay be true solvates, while in other cases, the compound of thedisclosure may merely retain adventitious water or be a mixture of waterplus some adventitious solvent.

The term “oxysterol” as used herein is meant to encompass one or moreforms of oxidized cholesterol. The oxysterols described herein areeither independently or collectively active to bone growth in a patient,as described in WO 2013169399 A1, which is hereby incorporated byreference in its entirety.

The oxysterol, sterol or diol can be in a pharmaceutically acceptablesalt. Some examples of potentially pharmaceutically acceptable saltsinclude those salt-forming acids and bases that do not substantiallyincrease the toxicity of a compound, such as, salts of alkali metalssuch as magnesium, potassium and ammonium, salts of mineral acids suchas hydrochloride, hydriodic, hydrobromic, phosphoric, metaphosphoric,nitric and sulfuric acids, as well as salts of organic acids such astartaric, acetic, citric, malic, benzoic, glycollic, gluconic, gulonic,succinic, arylsulfonic, e.g., p-toluenesulfonic acids, or the like.

Pharmaceutically acceptable salts of oxysterol, sterol or diol includesalts prepared from pharmaceutically acceptable non-toxic bases or acidsincluding inorganic or organic bases, inorganic or organic acids andfatty acids. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethyl amine, tripropylamine,tromethamine, and the like. When the compound of the current applicationis basic, salts may be prepared from pharmaceutically acceptablenon-toxic acids, including inorganic and organic acids. Such acidsinclude acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Fatty acid salts may also beused, eg., fatty acid salts having greater than 2 carbons, greater than8 carbons or greater than 16 carbons, such as butyric, caproic,caprylic, capric, lauric, mystiric, palmitic, stearic, arachidic or thelike.

In some embodiments, in order to reduce the solubility of the oxysterol,sterol, or diol to assist in obtaining a controlled release depoteffect, the oxysterol, sterol, or diol is utilized as the free base orutilized in a salt which has relatively lower solubility. For example,the present application can utilize an insoluble salt such as a fattyacid salt. Representative fatty acid salts include salts of oleic acid,linoleic acid, or fatty acid salts with between 8 to 20 carbonssolubility, such as for example, palmeate or stearate.

The term “solvate” is a complex or aggregate formed by one or moremolecules of a solute, e.g. a compound or a pharmaceutically-acceptablesalt thereof, and one or more molecules of a solvent. Such solvates canbe crystalline solids having a substantially fixed molar ratio of soluteand solvent. Suitable solvents will be known by those of ordinary skillin the art, e.g., water, ethanol.

The terms “bioactive” composition or “pharmaceutical” composition asused herein may be used interchangeably. Both terms refer tocompositions that can be administered to a subject. Bioactive orpharmaceutical compositions are sometimes referred to herein as“pharmaceutical compositions” or “bioactive compositions” of the currentdisclosure. Sometimes the phrase “administration of Oxy133” is usedherein in the context of administration of this compound to a subject(e.g., contacting the subject with the compound, injecting the compound,administering the compound in a drug depot, etc.). It is to beunderstood that the compound for such a use can generally be in the formof a pharmaceutical composition or bioactive composition comprising theOxy133.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the oxysterol (e.g., Oxy133), sterol, diol, resultsin alteration of the biological activity, such as, for example,enhancing bone growth, etc. The dosage administered to a patient can beas single or multiple doses depending upon a variety of factors,including the drug's administered pharmacokinetic properties, the routeof administration, patient conditions and characteristics (sex, age,body weight, health, size, etc.), and extent of symptoms, concurrenttreatments, frequency of treatment and the effect desired. In someembodiments the formulation is designed for immediate release. In otherembodiments the formulation is designed for sustained release. In otherembodiments, the formulation comprises one or more immediate releasesurfaces and one or more sustained release surfaces.

A “depot” includes but is not limited to capsules, microspheres,microparticles, microcapsules, microfibers particles, nanospheres,nanoparticles, coating, matrices, wafers, pills, pellets, emulsions,liposomes, micelles, gels, or other pharmaceutical delivery compositionsor a combination thereof. Suitable materials for the depot are ideallypharmaceutically acceptable biodegradable and/or any bioabsorbablematerials that are preferably FDA approved or GRAS materials. Thesematerials can be polymeric or non-polymeric, as well as synthetic ornaturally occurring, or a combination thereof.

The term “implantable” as utilized herein refers to a biocompatibledevice (e.g., drug depot) retaining potential for successful placementwithin a mammal. The expression “implantable device” and expressions ofthe like import as utilized herein refers to an object implantablethrough surgery, injection, or other suitable means whose primaryfunction is achieved either through its physical presence or mechanicalproperties.

“Localized” delivery includes delivery where one or more drugs aredeposited within a tissue, for example, a bone cavity, or in closeproximity (within about 0.1 cm, or preferably within about 10 cm, forexample) thereto. For example, the drug dose delivered locally from thedrug depot may be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 99.9% or 99.999% less than the oral dosage or injectabledose.

The term “mammal” refers to organisms from the taxonomy class“mammalian,” including but not limited to humans, other primates such aschimpanzees, apes, orangutans and monkeys, rats, mice, cats, dogs, cows,horses, etc.

The oxysterol can be “osteogenic,” where it can enhance or acceleratethe ingrowth of new bone tissue by one or more mechanisms such asosteogenesis, osteoconduction and/or osteoinduction.

New compositions and methods are provided to efficiently and safely makeoxysterols including Oxy133. Methods and compositions that canefficiently and safely generate Oxy133 are also provided.

The section headings below should not be restricted and can beinterchanged with other section headings.

Oxysterols

The present disclosure includes an osteogenic oxysterol (e.g., Oxy133),sterol, or diol and its ability to promote osteogenic differentiation invitro. Oxy133 is a particularly effective osteogenic agent. In variousapplications, Oxy133 is useful in treating conditions that would benefitfrom localized stimulation of bone formation, such as, for example,spinal fusion, fracture repair, bone regenerative/tissue applications,augmentation of bone density in the jaw for dental implants,osteoporosis or the like. One particular advantage of Oxy133 is that itprovides greater ease of synthesis and improved time to fusion whencompared to other osteogenic oxysterols. Oxy133 is a small molecule thatcan serve as an anabolic therapeutic agent for bone growth, as well as auseful agent for treatment of a variety of other conditions.

One aspect of the application disclosure is a compound, named Oxy133,having the formula:

or a pharmaceutically acceptable salt, solvate or hydrate thereof. TheOxy133 may be used as a bioactive or pharmaceutical compositioncomprising Oxy133 or a pharmaceutically acceptable salt, solvate orhydrate thereof and a pharmaceutically acceptable carrier.

Another aspect of the disclosure is a method for inducing (stimulating,enhancing) a hedgehog (Hh) pathway mediated response, in a cell ortissue, comprising contacting the cell or tissue with a therapeuticallyeffective amount of Oxy133. The cell or tissue can be in vitro or in asubject, such as a mammal. The hedgehog (Hh) pathway mediated responseinvolves the stimulation of osteoblastic differentiation,osteomorphogenesis, and/or osteoproliferation; the stimulation of hairgrowth and/or cartilage formation; the stimulation of neovasculogenesis,e.g. angiogenesis, thereby enhancing blood supply to ischemic tissues;or it is the inhibition of adipocyte differentiation, adipocytemorphogenesis, and/or adipocyte proliferation; or the stimulation ofprogenitor cells to undergo neurogenesis. The Hh mediated response cancomprise the regeneration of any of a variety of types of tissues, foruse in regenerative medicine. Another aspect of the disclosure is amethod for treating a subject having a bone disorder, osteopenia,osteoporosis, or a bone fracture, comprising administering to thesubject an effective amount of a bioactive composition or pharmaceuticalcomposition comprising Oxy133. The subject can be administered thebioactive composition or pharmaceutical composition at a therapeuticallyeffective dose in an effective dosage form at a selected interval to,e.g., increase bone mass, ameliorate symptoms of osteoporosis, reduce,eliminate, prevent or treat atherosclerotic lesions, or the like. Thesubject can be administered the bioactive composition or pharmaceuticalcomposition at a therapeutically effective dose in an effective dosageform at a selected interval to ameliorate the symptoms of osteoporosis.In some embodiments, a composition comprising Oxy133 may includemesenchymal stem cells to induce osteoblastic differentiation of thecells at a targeted surgical area.

In various aspects, the Oxy133 can be administered to a cell, tissue ororgan by local administration. For example, the Oxy133 can be appliedlocally with a cream or the like, or it can be injected or otherwiseintroduced directly into a cell, tissue or organ, or it can beintroduced with a suitable medical device, such as a drug depot asdiscussed herein.

In some embodiments, the dosage of Oxy133, sterol, or diol is fromapproximately 10 pg/day to approximately 80 mg/day. Additional dosagesof Oxy133, sterol, or diol include from approximately 2.4 ng/day toapproximately 50 mg/day; approximately 50 ng/day to approximately 2.5mg/day; approximately 250 ng/day to approximately 250 mcg/day;approximately 250 ng/day to approximately 50 mcg/day; approximately 250ng/day to approximately 25 mcg/day; approximately 250 ng/day toapproximately 1 mcg/day; approximately 300 ng/day to approximately 750ng/day or approximately 0.50 mcg/day to 500 ng/day. In variousembodiments, the dose may be about 0.01 to approximately 10 mcg/day orapproximately 1 ng/day to about 120 mcg/day.

In addition to the compound Oxy133, sterol, or diol other embodiments ofthe disclosure encompass any and all individual stereoisomers at any ofthe stereocenters present in Oxy133, including diastereomers, racemates,enantiomers, and other isomers of the compound. In embodiments of thedisclosure, Oxy133, sterol, oxysterol, diol may include all polymorphs,solvates or hydrates of the compound, such as hydrates and those formedwith organic solvents.

The ability to prepare salts depends on the acidity or basicity of acompound. Suitable salts of the compound include, but are not limitedto, acid addition salts, such as those made with hydrochloric,hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, benzoic, carbonic cinnamic, mandelic,methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; saltsmade with saccharin; alkali metal salts, such as sodium and potassiumsalts; alkaline earth metal salts, such as calcium and magnesium salts;and salts formed with organic or inorganic ligands, such as quaternaryammonium salts. Additional suitable salts include, but are not limitedto, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate,tosylate, triethiodide and valerate salts of the compounds.

In various embodiments, Oxy133, sterol, or diol includes one or morebiological functions. That is, Oxy133, sterol, or diol can induce abiological response when contacted with a mesenchymal stem cell or abone marrow stromal cell. For example, Oxy133, sterol, or diol maystimulate osteoblastic differentiation. In some embodiments, a bioactivecomposition including Oxy133 sterol, or diol may include one or morebiological functions when administered to a mammalian cell, for example,a cell in vitro or a cell in a human or an animal. For example, such abioactive composition may stimulate osteoblastic differentiation. Insome embodiments, such a biological function can arise from stimulationof the hedgehog pathway.

Methods of Making Intermediary Diol

In some embodiments, the current disclosure provides a method for thepreparation of an intermediary diol used in the production of Oxy133, asshown below. The diol may be used to promote bone growth as well.Previous methods of synthesis for Oxy133 produce were inefficient andnot suitable for scale up manufacturing. Some stereoisomers of Oxy133perform less optimally than others. The disclosed method isstereoselective and produces a high yield of the specific isomeric formof the diol shown below, which has been shown to produce an optimallyeffective isomeric form of Oxy133.

Disclosed are multiple embodiments of reactions to synthesize theintermediary diol. The diol synthesized has the IUPAC designation(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.Generally, the method of synthesizing the diol includes reactingpregnenolone, pregnenolone acetate or a pregnenolone derivative with anorganometallic reagent to facilitate alkylation of the C17 position, asshown below:

In one embodiment, as shown above in scheme 1, pregnenolone acetate(formula 1) may be alkylated by an organometallic reagent to synthesizethe intermediary diol, shown above as formula 2. In some embodiments,pregnenolone acetate is reacted with a Grignard reagent to facilitatealkylation of the C17 position on the pregnenolone acetate molecule. Insome embodiments, n-hexylmagnesium chloride is used as theorganometallic reagent.

In some embodiments, as shown above as scheme 2, pregnenolone is reactedwith a Grignard reagent such as n-hexylmagnesium chloride to facilitatealkylation of the C17 position of the pregnenolone molecule to form theintermediary diol shown as formula 2.

The method of synthesizing the intermediary diol (formula 2) or(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-olis stereoselective and produces a high yield of the diol. For example,in some embodiments, the yield of the desired stereoisomer of the diolis between about 60% and about 70%. In some embodiments, the yield ofthe desired stereoisomer of the diol is between about 50% and about 60%.However, it is contemplated that the percent yield may be higher orlower than these amounts. For example, the percent yield of formula 2 asshown above may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or 95%. In some embodiments, the percentyield may be above 95%.

In various embodiments, the alkylation reaction is carried out in apolar organic solvent, such as tetrahydrofuran. However, the reactionmay be carried out in a variety of polar organic solvents. For example,the reaction may be carried out in diethyl ether, ethyl ether, dimethylether or the like.

In some embodiments, pregnenolone or pregnenolone acetate is used as astarting reactant. However, in other embodiments, derivatives ofpregnenolone acetate may be used. For example, other specific examplesof compounds which could be used in the present disclosure include:pregnenolone sulfate, pregnenolone phosphate, pregnenolone formate,pregnenolone hemioxalate, pregnenolone hemimalonate, pregnenolonehemiglutarate, 20-oxopregn-5-en-3β-yl carboxymethyl ether,3β-hydroxypregn-5-en-20-one sulfate,3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one,3-hydroxy-19-norpregna-1,3,5(10),6,8-pentaen-20-one, 17α-isopregnenolonesulfate, 17-acetoxypregnenolone sulfate, 21-hydroxypregnenolone sulfate,20β-acetoxy-3β-hydroxypregn-5-ene-sulfate, pregnenolone sulfate20-ethyleneketal, pregnenolone sulfate 20-carboxymethyloxime,20-deoxypregnenolone sulfate, 21-acetoxy-17-hydroxypregnenolone sulfate,17-propyloxypregnenolone sulfate, 17-butyloxypregnenolone sulfate,21-thiol esters of pregnenolone sulfate, pyridinium, imidazolium,6-methylpregnenolone sulfate, 6,16α-dimethylpregnenolone sulfate,3β-hydroxy-6-methylpregna-5,16-dien-20-one sulfate,3β-hydroxy-6,16-dimethylpregna-5,16-dien-20-one sulfate,3jβ-hydroxypregna-5,16-dien-20-one sulfate, diosgenin sulfate,3β-hydroxyandrost-5-en-17β-carboxylic acid methyl ester sulfate, 3αhydroxy-5β-pregnan-20-one formate, 3α-hydroxy-5β-pregnan-20-onehemioxalate, 3α-hydroxy-5β-pregnan-20-one hemimalonate,3α-hydroxy-5β-pregnan-20-one hemisuccinate, 3α-hydroxy-5β-pregnan-20-onehemiglutarate, estradiol-3-formate, estradiol-3-hemioxalate,estradiol-3-hemimalonate, estradiol-3-hemisuccinate,estradiol-3-hemiglutarate, estradiol-17-methyl ether,estradiol-17-formate, estradiol-17-hemioxalate,estradiol-17-hemimalonate, estradiol-17-hemisuccinate,estradiol-17-hemiglutarate, estradiol-3-methyl ether, 17-deoxyestrone,and 17β-hydroxyestra-1,3,5(10)-trien-3-yl carboxymethyl ether.

In some embodiments, the organometallic comprises n-hexylmagnesiumchloride. However, in some embodiments, the alkylation reaction may becarried out with the use of an alkyllithium, such as, for example,n-hexyllithium. In various embodiments, the organometallic includes analkyl halide. For example, the organometallic reagent may have thefollowing formula:

R—Mg—X,

where Mg comprises magnesium, X comprises chlorine, bromine, fluorine,iodine, or astatine and R comprises an alkyl, a heteroalkyl, an alkanyl,a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, aheteroalkanyl, an alkyldiyl, a heteroalkyldiyl, an alkyleno, aheteroalkyleno, an aryl, an aryldiyl, an arydeno, an arylaryl, a biaryl,an arylalkyl, a heteroaryl, a heteroaryldiyl, a heteroaryleno, aheteroaryl-heteroaryl, a biheteroaryl, a heteroarylalkyl or combinationsthereof. In some embodiments, the R substituent comprises a (C1-C20)alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno or heteroalkyleno. The R substituent may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted, aromatic,saturated or unsaturated chains, or combinations thereof. In someembodiments, the R substituent is an aliphatic group. In someembodiments, the R substituent is a cyclic group. In some embodiments,the R substituent is a hexyl group.

Alternatively, the organometallic may comprise the formula:

R—Li,

where Li comprises lithium and R comprises an alkyl, a heteroalkyl, analkanyl, a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, aheteroalkanyl, an alkyldiyl, a heteroalkyldiyl, an alkyleno, aheteroalkyleno, an aryl, an aryldiyl, an arydeno, an arylaryl, a biaryl,an arylalkyl, a heteroaryl, a heteroaryldiyl, a heteroaryleno, aheteroaryl-heteroaryl, a biheteroaryl, a heteroarylalkyl or combinationsthereof. In some embodiments, the R substituent comprises a (C₁-C₂₀)alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno or heteroalkyleno. The R substituent may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted, aromatic,saturated or unsaturated chains, or combinations thereof. In someembodiments, the R substituent is an aliphatic group. In someembodiments, the R substituent is a cyclic group. In some embodiments,the R substituent is a hexyl group.

In some embodiments, the alkylation reaction is exothermic and thereaction vessel may be temperature controlled to maintain optimalreaction kinetics. In some embodiments, the exothermic reaction releasesabout 1000 BTU per pound of solution. Due to the strongly exothermicnature of the reaction, the Grignard reagent therefore must be addedslowly so that volatile components, for example ethers, are notvaporized due to the reaction heat. In some embodiments, the reactionvessel may be cooled by internal cooling coils. The cooling coils may besupplied with a coolant by means of an external gas/liquid refrigerationunit. In some embodiments, an internal temperature of the reactionvessel is maintained at less than 15° C., 10° C., 5° C. or 1° C. In someembodiments, the reaction vessel is maintained at about 0° C. during thealkylation reaction to form the intermediary diol of formula 2.

In various embodiments, the diol of formula 2 is synthesized along withbyproducts and can be purified. For example, the resulting diol offormula 2 may be a byproduct of a diastereomeric mixture. In variousembodiments, the diol of formula 2 may be isolated and purified. Thatis, the diol of formula 2 can be isolated and purified to the desiredpurity, e.g., from about 95% to about 99.9% by filtration,centrifugation, distillation, which separates volatile liquids on thebasis of their relative volatilities, crystallization,recrystallization, evaporation to remove volatile liquids fromnon-volatile solutes, solvent extraction to remove impurities,dissolving the composition in a solvent in which other components aresoluble therein or other purification methods. The diol may be purifiedby contacting it with organic and/or inorganic solvents, for example,THF, water, diethyl ether, dichloromethane, ethyl acetate, acetone,n,n-dimethylformamide, acetonitrile, dimethyl sulfoxide, ammonia,t-butanol, n-propanol, ethanol, methanol, acetic acid, or a combinationthereof.

In various embodiments, the alkylation step and the purification steptake place in the same reaction vessel.

In some embodiments, the diol is quenched with aqueous ammonium chlorideor acetic acid to reduce the amount of anions present and neutralize thereaction and separated from the resulting organic layer. The separatedresidue is recovered by evaporation and purified by silica gel columnchromatography.

The diol may be anhydrous or in the monohydrate form. However, in otherembodiments the purified diol may be crystallized in other hydrousforms, such as, for example, a dihydrate, a hemihydrate, asesquihydrate, a trihydrate, a tetrahydrate and the like, as well as thecorresponding solvated forms. In other embodiments, the purified diol iscrystallized as a co-crystal or a pharmaceutically acceptable salt.

Methods of Making Oxy133

In some embodiments, the current disclosure provides a method for thepreparation of an Oxy133, as shown below. Previous methods of synthesisfor Oxy133 produce diastereomeric mixtures of Oxy133 intermediates whichrequire purification methods to separate. As discussed above to form theintermediary diol, the disclosed method is stereoselective and producesa high yield of the specific isomeric forms of Oxy133. The formula ofOxy133 is shown below.

Disclosed are multiple embodiments of reactions to synthesize Oxy133.Oxy133 has the IUPAC designation(3S,5S,6S,8R,9S,10R,13S,14S,17S)-17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol.Oxy133 has previously been synthesized through a complex process notsuitable for scale-up as shown below:

However, the reaction has difficulty being carried out in a singlecontainer. The reaction shown above involves more reagents to carry outreaction steps (e.g., blocking and deprotection groups and steps) whichhave an adverse environmental impact. Additionally, the known methodsinvolve reagents that are expensive and often difficult to obtain.Further, the method shown in Scheme 3 gives relatively low yields, hasmore degradation products, impurities and creates many toxic byproducts.

Generally, the method of synthesizing Oxy133 as disclosed hereinincludes reacting the diol synthesized as described herein with boranein the reaction shown below:

In some embodiments, crude and unpurified Oxy133 is produced through ahydroboration and oxidation reaction of the intermediary diol havingformula 2 in reaction scheme 4. Borane compounds that can be used in thereaction include BH₃, B₂H₆, BH₃S(CH₃)₂ (BMS), borane adducts withphosphines and amines, e.g., borane triethylamine; monosubstitutedboranes of the form RBH₂ where R=alkyl and halide, monoalkyl boranes(e.g., IpcBH2, monoisopinocampheylborane), monobromo- andmonochloro-borane, complexes of monochloroborane and 1,4-dioxane,disubstituted boranes including bulky boranes, such as for example,dialkylborane compounds such as diethylborane,bis-3-methyl-2-butylborane (disiamylborane), 9-borabycyclo[3,3,1]nonane(9-BBN), disiamylborane (Sia2BH), dicyclohexylborane, Chx2BH,trialkylboranes, dialkylhalogenoboranes, dimesitylborane (C₆H₂Me₃)₂BH,alkenylboranes, pinacolborane, or catecholborane or a combinationthereof.

Briefly, a hydroboration and oxidation reaction is a two-step reaction.The boron and hydrogen add across the double bond of an alkene to form acomplex with the alkene. Thus the boration phase of the reaction isstereoselective and regioselective. The oxidation phase of the reactioninvolves basic aqueous hydrogen peroxide to furnish a hydroxylsubstituent in place of the boron. See Vollhart, K P, Schore, N E, 2007,Organic Chemistry: Structure and Function, Fifth Ed., New York, N.Y.,Custom Publishing Company. Thus, the intermediary diol having formula 2is reacted with borane and hydrogen peroxide to form crude Oxy133. Insome embodiments, the step of forming crude Oxy133 takes place in thesame reaction vessel as the alkylation reaction. In other embodiments,the step of forming crude Oxy133 takes place in a different reactionvessel as the alkylation reaction.

The hydroboration-oxidation step of the synthesis of Oxy133, like thestep of forming the intermediary diol, is stereoselective and produces ahigh yield. For example, in some embodiments, the percent yield of crudeOxy133 may be higher or lower than these amounts. For example, thepercent yield of formula 2 as shown above may be about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Insome embodiments, the percent yield may be above 95%.

In various embodiments, the hydroboration-oxidation reaction is carriedout in a polar organic solvent, such as tetrahydrofuran. However, thereaction may be carried out in a variety of polar organic solvents. Forexample, the reaction may be carried out in diethyl ether, ethyl ether,dimethyl ether or the like.

In some embodiments, the hydroboration-oxidation reaction is exothermicand the reaction vessel must be temperature controlled to maintainoptimal reaction kinetics. Specifically, the oxidation phase isextremely exothermic. Due to the strongly exothermic nature of thereaction, the hydrogen peroxide therefore can be added slowly so thatvolatile components, for example ethers, are not vaporized due to thereaction heat. In some embodiments, the reaction vessel may be cooled byinternal cooling coils. The cooling coils may be supplied with a coolantby means of an external gas/liquid refrigeration unit. In someembodiments, an internal temperature of the reaction vessel ismaintained at less than 10° C., 5° C., 1° C. or 0° C. In someembodiments, the reaction vessel is maintained at about −5° C. duringthe hydroboration-oxidation reaction.

In certain embodiments the diol can have a percent crystallinity of asalt, hydrate, solvate or crystalline form of diol to be at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least, 60%,at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.In some embodiments, the percent crystallinity can be substantially100%, where substantially 100% indicates that the entire amount of diolappears to be crystalline as best can be determined using methods knownin the art. Accordingly, therapeutically effective amounts of diol caninclude amounts that vary in crystallinity. These include instanceswhere an amount of the crystallized diol in a solid form is subsequentlydissolved, partially dissolved, or suspended or dispersed in a liquid.

Purification of Oxy133

In some embodiments, the crude Oxy133 must be separated from thereaction mixture prior to purification. In some embodiments, an organicsolvent such as dichloromethane is added to the crude Oxy133 reactionmixture and the resulting organic layer is separated. Once separated,the crude Oxy133 exists as a semi-solid viscous mass. The crude Oxy133may be dissolved by any suitable means (e.g., dichloromethane, etc.) andplaced into a silica gel column with an organic solvent, such asmethanol-ethyl acetate, to solvate the crude Oxy133. In someembodiments, the crude Oxy133 may be crystallized or recrystallized. Insome embodiments, purified Oxy133 is formed by recrystallizing the crudeOxy133 in a 3:1 mixture of acetone/water, as shown below:

As shown above, upon crystallization, the purified Oxy133 forms ahydrate. However, it can be in the anhydrous form. In some embodiments,the percent crystallinity of any of the crystalline forms of Oxy133described herein can vary with respect to the total amount of Oxy133.

In certain embodiments the Oxy133 can have a percent crystallinity of asalt, hydrate, solvate or crystalline form of Oxy133 to be at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least, 60%,at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.In some embodiments, the percent crystallinity can be substantially100%, where substantially 100% indicates that the entire amount ofOxy133 appears to be crystalline as best can be determined using methodsknown in the art. Accordingly, therapeutically effective amounts ofOxy133 can include amounts that vary in crystallinity. These includeinstances where an amount of the crystallized Oxy133 in a solid form issubsequently dissolved, partially dissolved, or suspended or dispersedin a liquid.

In one embodiment, the purified Oxy133 is crystallized as a monohydrate.However, in other embodiments the purified Oxy133 may be crystallized inother hydrous forms, such as, for example, a dihydrate, a hemihydrate, asesquihydrate, a trihydrate, a tetrahydrate and the like, as well as thecorresponding solvated forms. In other embodiments, the purified Oxy133is crystallized as a co-crystal or a pharmaceutically acceptable salt.

In some embodiments, the reaction mixture containing the crude Oxy133may be solidified by mixing with heptanes. The product may subsequentlybe filtered and suspended in methylene chloride. In some embodiments,the crude Oxy133 may be filtered from the suspension and crystallizedwith the use of acetone and water or other organic or inorganic solvents(e.g., diethyl ether, dichloromethane, ethyl acetate, acetone,n,n-dimethylformamide, acetonitrile, dimethyl sulfoxide, ammonia,t-butanol, n-propanol, ethanol, methanol, acetic acid or a combinationthereof).

In various embodiments, the crude Oxy133 may be isolated and purified byany other traditional means. That is, the crude Oxy133 can be isolatedand purified to the desired purity, e.g., from about 95% to about 99.9%by filtration, centrifugation, distillation to separate volatile liquidson the basis of their relative volatilities, crystallization,recrystallization, evaporation to remove volatile liquids fromnon-volatile solutes, solvent extraction to remove impurities,dissolving the composition in a solvent in which other components aresoluble therein or other purification methods. In various embodiments,the hydroboration-oxidation step and the purification step take place inthe same reaction vessel. In various embodiments, the alkylation step,the hydroboration-oxidation step and the purification step take place inthe same reaction vessel.

The method of synthesizing the intermediary diol (formula 2) is stereoselective and produces a high yield of Oxy133. For example, in someembodiments, the yield of the purified Oxy133 is between about 20% andabout 99%. In some embodiments, the yield of the purified Oxy133 isbetween about 20% and about 80%. In some embodiments, the yield of thepurified Oxy133 is between about 25% and about 70% or about 28%.However, it is contemplated that the percent yield may be higher orlower than these amounts.

In some embodiments, the purified Oxy133 is formed in crystal form viacrystallization, which separates the Oxy133 from the liquid feed streamby cooling the liquid feed stream or adding precipitants which lower thesolubility of byproducts and unused reactants in the reaction mixture sothat the Oxy133 forms crystals. In some embodiments, the solid crystalsare then separated from the remaining liquor by filtration orcentrifugation. The crystals can be resolubilized in a solvent and thenrecrystallized and the crystals are then separated from the remainingliquor by filtration or centrifugation to obtain a highly pure sample ofOxy133. In some embodiments, the crystals can then be granulated to thedesired particle size.

In some embodiments, the purity of the Oxy133 obtained is verifiedthrough nuclear magnetic resonance or mass spectroscopy. As shown inFIGS. 2-5, 1H NMR, 13C NMR, infrared spectroscopy, and mass spectroscopyanalysis indicated that the Oxy133 product had high purity (e.g., having98% to about 99.99% by weight purity).

In some embodiments, the crude Oxy133 can be purified where the purifiedOxy133 is formed in crystalized form in a solvent and then removed fromthe solvent to form a high purity Oxy133 having a purity of from about98% to about 99.99%. In some embodiments, the Oxy133 can be recoveredvia filtration or vacuum filtration before or after purification.

Use of Oxysterols

In use, Oxy133 provides therapeutic treatment for bone conditions.Oxy133 facilitates bone formation, osteoblastic differentiation,osteomorphogenesis and/or osteoproliferation. Treatment can beadministered to treat open fractures and fractures at high risk ofnon-union, and in subjects with spinal disorders. That is, Oxy133 caninduce spinal fusion and may help treat degenerative disc disease orarthritis affecting the lumbar or cervical vertebrae.

Mesenchymal stem cells treated with Oxy133 have been shown to haveincreased osteoblast differentiation. Thus, in some embodiments, Oxy133may be implanted into a spinal site with mesenchymal stem cells toinduce bone growth through osteoblast differentiation. Periosteum tissueis one tissue type that is involved early during normal bone fracturerepair process and can recruit various cell types (e.g., mesenchymalstem cells) and bone growth factors necessary for bone fracture repair.Thus, in some embodiments, periosteum tissue is utilized as a source ofmesenchymal stem cells and/or growth factors in a demineralized bonecomposition.

In some embodiments, the Oxy133 may be implanted or injected directly toa surgical site on a patient. In some embodiments, the Oxy133 obtainedfrom the methods delineated above is in the form of a depot. In variousembodiments, a plurality of depots (e.g., pellets) can be administeredto a surgical site. In some embodiments, a plurality of depots areprovided (e.g., in a kit) and administered to a surgical site andtriangulate and/or surround the site needed for bone growth. In variousembodiments, a plurality of depots comprise about 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 depots. In some embodiments, a plasticizer is used to lowerglass translation temperature in order to affect stability of thedevice.

In various embodiments, the depot comprises Oxy133, sterol, or diol anda biodegradable polymer in amorphous, crystalline or semicrystallineform; where the crystalline form may include polymorphs, solvates orhydrates.

In some embodiments, Oxy133, sterol, or diol is administered in a devicethat is solid or in semi-solid form. The solid or semi-solid form of thedevice may have a pre-dosed viscosity in the range of about 1 to about2000 centipoise (cps), 1 to about 200 cps, or 1 to about 100 cps. Afterthe solid or semi-solid device is administered to the target site, theviscosity of the semi-solid or solid depot will increase and thesemi-solid will have a modulus of elasticity in the range of about 1×10²to about 6×10⁵ dynes/cm², or 2×10⁴ to about 5×10⁵ dynes/cm², or 5×10⁴ toabout 5×10⁵ dynes/cm².

In various embodiments, the semi-solid or solid depot may comprise apolymer having a molecular weight (MW), as shown by the inherentviscosity, from about 0.10 dL/g to about 1.2 dL/g or from about 0.20dL/g to about 0.50 dL/g. Other IV ranges include but are not limited toabout 0.05 to about 0.15 dL/g, about 0.10 to about 0.20 dL/g, about 0.15to about 0.25 dL/g, about 0.20 to about 0.30 dL/g, about 0.25 to about0.35 dL/g, about 0.30 to about 0.35 dL/g, about 0.35 to about 0.45 dL/g,about 0.40 to about 0.45 dL/g, about 0.45 to about 0.55 dL/g, about 0.50to about 0.70 dL/g, about 0.55 to about 0.6 dL/g, about 0.60 to about0.80 dL/g, about 0.70 to about 0.90 dL/g, about 0.80 to about 1.00 dL/g,about 0.90 to about 1.10 dL/g, about 1.0 to about 1.2 dL/g, about 1.1 toabout 1.3 dL/g, about 1.2 to about 1.4 dL/g, about 1.3 to about 1.5dL/g, about 1.4 to about 1.6 dL/g, about 1.5 to about 1.7 dL/g, about1.6 to about 1.8 dL/g, about 1.7 to about 1.9 dL/g, or about 1.8 toabout 2.1 dL/g.

In some embodiments, the depot may not be fully biodegradable. Forexample, the device may comprise polyurethane, polyurea,polyether(amide), PEBA, thermoplastic elastomeric olefin, copolyester,and styrenic thermoplastic elastomer, steel, aluminum, stainless steel,titanium, metal alloys with high non-ferrous metal content and a lowrelative proportion of iron, carbon device, glass device, plastics,ceramics, methacrylates, poly (N-isopropylacrylamide), PEO-PPO-PEO(pluronics) or combinations thereof. Typically, these types of matricesmay need to be removed after a certain amount of time.

In various embodiments, the depot (e.g., device) may comprise abioerodible, a bioabsorbable, and/or a biodegradable biopolymer that mayprovide immediate release, or sustained release of the Oxy133. Examplesof suitable sustained release biopolymers include but are not limited topoly (alpha-hydroxy acids), poly (lactide-co-glycolide) (PLGA),polylactide (PLA), polyglycolide (PG), polyethylene glycol (PEG)conjugates of poly (alpha-hydroxy acids), poly(orthoester)s (POE),poly(esteramide)s, polyaspirins, polyphosphagenes, starch,pre-gelatinized starch, hyaluronic acid, chitosans, gelatin, alginates,albumin, fibrin, vitamin E compounds, such as alpha tocopheryl acetate,d-alpha tocopheryl succinate, D,L-lactide, or L-lactide-caprolactone,dextrans, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA,PEGT-PBT copolymer (polyactive), PEO-PPO-PAA copolymers, PLGA-PEO-PLGA,PEG-PLG, PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblock copolymers, SAIB(sucrose acetate isobutyrate) or combinations thereof.

In some embodiments, the depot comprises biodegradable polymerscomprising wherein the at least one biodegradable polymer comprises oneor more of poly(lactide-co-glycolide) (PLGA), polylactide (PLA),polyglycolide (PGA), D-lactide, D,L-lactide, L-lactide,D,L-lactide-co-ε-caprolactone, L-lactide-co-ε-caprolactone,D,L-lactide-co-glycolide-co-ε-caprolactone,poly(D,L-lactide-co-caprolactone), poly(L-lactide-co-caprolactone),poly(D-lactide-co-caprolactone), poly(D,L-lactide), poly(D-lactide),poly(L-lactide), poly(esteramide) or a combination thereof.

In some embodiments, the depot comprises at least one biodegradablematerial in a wt % of about 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%,78%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, 60%, 55%, 50%, 45%, 35%,25%, 20%, 15%, 10%, or 5% based on the total weight of the depot and theremainder is active and/or inactive pharmaceutical ingredients.

Mannitol, trehalose, dextran, mPEG and/or PEG may be used as aplasticizer for the polymer. In some embodiments, the polymer and/orplasticizer may also be coated on the depot to provide the desiredrelease profile. In some embodiments, the coating thickness may be thin,for example, from about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 micronsto thicker coatings 60, 65, 70, 75, 80, 85, 90, 95, 100 microns to delayrelease of the Oxy133, sterol, or diol from the depot (e.g., device). Insome embodiments, the range of the coating on the depot ranges fromabout 5 microns to about 250 microns or 5 microns to about 200 micronsto delay release from the device.

The depot (e.g., device) can be different sizes, shapes andconfigurations. There are several factors that can be taken intoconsideration in determining the size, shape and configuration of thedepot. For example, both the size and shape may allow for ease inpositioning the depot at the target tissue site that is selected as theimplantation. In addition, the shape and size of the system should beselected so as to minimize or prevent the depot from moving afterimplantation. In various embodiments, the depot can be shaped like a rodor a flat surface such as a film or sheet (e.g., ribbon-like) or thelike. Flexibility may be a consideration so as to facilitate placementof the device.

Radiographic markers can be included on the device to permit the user toposition the depot (e.g., device) accurately into the target site of thepatient. These radiographic markers will also permit the user to trackmovement and degradation of the depot (e.g., device) at the site overtime. In this embodiment, the user may accurately position the depot(e.g., device) in the site using any of the numerous diagnostic imagingprocedures. Such diagnostic imaging procedures include, for example,X-ray imaging or fluoroscopy. Examples of such radiographic markersinclude, but are not limited to, barium, phosphate, bismuth, iodine,tantalum, tungsten, and/or metal beads or particles. In variousembodiments, the radiographic marker could be a spherical shape or aring around the depot (e.g., device).

In some embodiments, the Oxy133, sterol, or diol can be administered tothe target site using a “cannula” or “needle” that can be a part of adelivery device e.g., a syringe, a gun delivery device, or any medicaldevice suitable for the application of Oxy133, sterol, or diol to atargeted organ or anatomic region. The cannula or needle of the deviceis designed to cause minimal physical and psychological trauma to thepatient.

In some embodiments, the depot can be sutured to a target tissue siteusing a suturing needle. The dimensions of the needle, among otherthings, will depend on the site for implantation. For example, the widthof the muscle planes in different surgical procedures can vary from 1-40cm. Thus, the needle, in various embodiments, can be designed for thesespecific areas.

These and other aspects of the present application will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the applicationbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Preparation from Pregnenolone Acetate

8.25 mL n-hexylmagnesium chloride (2 M, 16.5 mmol) in THF was added to asolution of pregnenolone acetate in THF under vigorous electromagneticstirring and ice bath cooling. The pregnenolone acetate solutioncontained 1.79 g compound 1, pregnenolone acetate, (5 mmol) in 4.5 mLTHF. The addition took place over 2 minutes. After addition wascompleted, the mixture was stirred at room temperature for 3.5 hours, atwhich point the mixture had turned to a gel. The gel was then digestedwith a mixture of saturated aqueous NH₄Cl and MTBE (methyltertiary-butyl ether). The organic layer was separated, washed withwater three times and evaporated. The residue was separated by silicagel column chromatography using an EtOAc (ethyl acetate)/petroleum ethermixture (ratio 70/30) to give compound 2, a diol, as a white solid. 1.29g (3.21 mmol) of the solid diol was extracted for a 64% isolated yield.The reaction is shown below in A:

The ¹H NMR data of the diol in CDCl₃ at 400 MHz illustrated thefollowing: δ: 0.8-1.9 (40H), 1.98 (m, 1H), 2.09 (m, 1H), 2.23 (m, 1H),2.29 (m, 1H), 3.52 (m, 1H), 5.35 (m, 1H) in FIG. 6. The ¹³C NMR data ofthe diol in CDCl₃ at 100 MHz in FIG. 7 illustrated the following: d:13.6, 14.1, 19.4, 20.9, 22.4, 22.6, 23.8, 24.2, 26.4, 30.0, 31.3, 31.6,31.8, 31.9, 36.5, 37.3, 40.1, 42.3, 42.6, 44.0, 50.1, 56.9, 57.6, 71.7,75.2, 121.6, 140.8.

The diol created has an IUPAC name of(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.

Example 2 Preparation from Pregnenolone

Alternatively to Example 1, compound 2 of reaction scheme A above can beprepared from pregnenolone shown below in B utilizing the same procedureas utilized for the conversion of compound 1 to compound 2. In thisprocedure 10 g of pregnenolone was converted to 7.05 g of compound 2,which accounted for a 55% yield.

2500 mL of n-hexylmagnesium chloride (2 M, 5 mol) was charged to areactor and the solution was cooled to −5° C. A solution of pregnenoloneacetate in THF was charged to the reactor at a rate which maintained theinternal reaction temperature below 1° C. The pregnenolone solutioncontained 500 g pregnenolone (1.4 mol) in 8 liters THF. After theaddition was complete, the mixture was held at 0° C. for 1 hour thenallowed to warm to room temperature overnight. The reaction mixture hadbecome a solid, gelatinous mass. 2 liters of additional THF was addedfollowed by 10 ml of glacial acetic acid. The reaction mixture wascooled to 5° C. and quenched by the addition of 350 ml of glacial aceticacid which gave a solution. The reaction mixture was concentrated underreduced pressure to a thick syrup. The compound was dissolved indichloromethane, washed with water and finally washed with saturatedsodium bicarbonate. The organic layer was concentrated under reducedpressure to an amber oil. Mass recovery was about 800 grams. The crudematerial was utilized as is in the next step.

The diol created has an IUPAC name of(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.

Example 3

The crude hexyl diol product (800 grams) was dissolved in 8 liters ofTHF, charged to a reactor, and was cooled to −5° C. 6300 mL ofborane-THF complex (1 M, 6.3 moles, 4.5 equivalents) in THF was chargedat a rate which maintained the internal reaction temperature below 1° C.Once the addition was complete, the reaction mixture was stirred at 0°C. for 1.5 hours then allowed to warm to room temperature overnight. Thereaction is shown below.

The reaction mixture was quenched by addition of a mixture of 10% sodiumhydroxide (4750 mL) and 30% hydrogen peroxide (1375 mL). The quench wasextremely exothermic and required several hours to complete. Theinternal temperature was maintained below 10° C. After the addition ofthe quench volume was complete, the mixture was held cold for 1.5 hoursthen allowed to warm to room temperature overnight. 8 liters ofdichloromethane was then added. The organic layer was isolated andwashed with 7 liters of fresh water, and was concentrated under reducedpressure. The product was isolated as a viscous, oily mass whichsolidified on standing.

The product was dissolved in 4 liters of dichloromethane and was placedonto a silica gel column prepared in dichloromethane. The column waseluted first with 25% ethyl acetate to elute the 7-methyl-7-tridecylalcohol by-product. Subsequently, the column was eluted with 10%methanol-ethyl acetate to solvate the Oxy133. The collected fractionswere combined and concentrated under reduced pressure to a waxy solid.The compound was dissolved in acetone-water mixture (3:1) andconcentrated under reduced pressure to remove residual solvents. Theresulting crude Oxy133 was utilized in the next step.

Alternatively, the viscous product recovered from thehydroboration/oxidation can be solidified by stirring with heptanes, andthe product isolated by filtration. The isolated product is suspended inmethylene chloride (7.3 mL methylene chloride/g solid). The product wasisolated by filtration and used as-is in the next step.

Example 4

Oxy133 was recrystallized by dissolving 630 grams of crude oxy133 into1500 ml of a 3:1 acetone/water mixture at reflux, then cooling to roomtemperature. The crystalline solid was recovered by vacuum filtrationand dried to afford 336 g, which was a 28% overall yield fromcompound 1. The Oxy133 produced was monohydrous, and has an IUPAC nameof(3S,5S,6S,8R,9S,10R,13S,14S,17S)-17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol,monohydrate.

The ¹H NMR data of Oxy133 in CDCl₃ at 400 MHz illustrated the following:δ: 0.66 (m, 1H), 0.85 (m, 10H), 1.23 (m, 18H), 1.47 (m, 9H), 1.68 (m,4H), 1.81 (m, 1H), 1.99 (m, 1H), 2.06 (m. 1H), 2.18 (m, 1H), 3.42 (m,1H), 3.58 (m, 1H). The ¹³C NMR data of Oxy133 in CDCl₃ at 400 MHzillustrated the following: d: 13.7, 14.0, 14.3, 21.2, 22.5, 22.8, 23.9,24.4, 26.6, 30.1, 31.1, 32.1, 32.5, 33.9, 36.5, 37.5, 40.4, 41.7, 43.1,44.3, 51.9, 53.9, 56.5, 57.9, 69.6, 71.3, 75.4. The infraredspectroscopy data of Oxy133 showed peaks at 3342 cm⁻¹, 2929 cm⁻¹, 2872cm⁻¹, 2849 cm⁻¹. The turbo spray mass spectrometry data of the Oxy133showed peaks at 438.4 m/z [M+NH₄]+, 420.4 m/z (M−H₂O+NH₄]+, 403.4 m/z[M−H₂O+H]+, 385.4 m/z [M−2H₂O+H]+. The ¹H NMR, ¹³C NMR, IR, and MS ofOxy133 data are shown in FIGS. 2, 3, 4 and 5, respectively.

Example 5 Alternative One-Vessel Procedure from Pregnenolone Acetate

100 mL n-hexylmagnesium chloride (2M in THF, 200 mmol) was charged to aflask and cooled to −10° C. A solution containing 20 g pregnenoloneacetate (56 mmol) in 200 ml of anhydrous THF) was added dropwise, whilemaintaining the internal reaction temperature below −10° C. After theaddition was completed, the mixture was stirred for 30 minutes thenallowed to warm to room temperature. After 4 hours at room temperature,the mixture had become a gelatinous stirrable mass. The mixture wascooled to 0° C. and 200 mL Borane-THF complex (1M in THF, 200 mmol) wasadded dropwise, while maintaining the internal temperature below 0° C.Once addition was complete, the resulting solution was allowed to warmto room temperature overnight.

The mixture was cooled to 0° C. and quenched by the slow addition of amixture of 10% NaOH (190 mL) and 30% H₂O₂ (55 mL). Once the quench wascomplete, the mixture was extracted with MTBE (800 mL total) resultingin an emulsion. Brine was added and the layers were separated. Theorganic phase was concentrated under reduced pressure to a clear,viscous oil. The oil was further purified utilizing the plug columnmethod previously described.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

What is claimed is:
 1. A method of making an oxysterol, the methodcomprising reacting a diol having the formula:

with a borane compound to form the oxysterol or a pharmaceuticallyacceptable salt, hydrate or solvate thereof having the formula:

wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom, and R2 comprises an aliphatic or cyclic substituenthaving at least one carbon atom.
 2. A method of claim 1, wherein theborane compound is BH₃ and is reacted with a peroxide.
 3. A method ofclaim 2, wherein the peroxide is hydrogen peroxide.
 4. A method of claim3, wherein the BH₃ and the hydrogen peroxide are reacted in the presenceof tetrahydrofuran to form the oxysterol.
 5. A method of claim 1,wherein the R1 and R2 comprise a hexyl group and the diol comprises(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-oland the hydrate is a monohydrate.
 6. A method of claim 4, wherein (i)the oxysterol is hydrated, (ii) the borane is reacted with the diol at atemperature of less than 5° C., (iii) the hydrogen peroxide is reactedwith the diol at a temperature of less than 15° C., or (iv) theoxysterol is solidified and separated by filtration.
 7. A method ofclaim 1, wherein the reaction is carried out in a single container toyield the oxysterol having the formula

or a pharmaceutically acceptable salt, hydrate or solvate thereof.
 8. Amethod of claim 1, wherein the oxysterol is purified by dissolving theoxysterol in an organic solvent.
 9. A method of claim 8, wherein theoxysterol is purified by dissolving the oxysterol in an acetone andwater to recover purified oxysterol crystals.
 10. A method of claim 8,wherein the oxysterol is purified by reflux and recrystallization bydissolving the oxysterol in an acetone and water to recover purifiedoxysterol crystals.
 11. A method of claim 1, wherein the oxysterol isanhydrous.
 12. A method of claim 10, wherein the purified oxysterol ishydrated after crystallization.
 13. A method of claim 10, wherein thepurified crystallized oxysterol is a monohydrate.
 14. A method of claim1, wherein the borane compound is reacted with the diol at a temperatureof less than 5° C.
 15. A method of claim 4, wherein the hydrogenperoxide is reacted with the diol after the borane is reacted and thehydrogen peroxide is reacted with the diol at a temperature of less than15° C.
 16. A method of claim 1, wherein the reaction is carried out in asingle container to yield the oxysterol.
 17. A method of making anoxysterol, the method comprising reacting a diol having the formula:

with borane, hydrogen peroxide and tetrahydrofuran to form the oxysterolor a pharmaceutically acceptable salt, hydrate or solvate thereof havingthe formula:

wherein R1 comprises an aliphatic or cyclic substituent having at leastone carbon atom, and R2 comprises an aliphatic or cyclic substituenthaving at least one carbon atom.
 18. A method of claim 17, wherein (i)the diol comprises(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-olor (ii) the oxysterol comprises3S,5S,6S,8R,9S,10R,13S,14S,17S)-17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol.19. A method of claim 17, wherein R₁ or R₂ comprises a (C₁-C₂₀) alkyl orheteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆) arylalkyl orheteroalkyl and a (C₅-C₂₀) arylalkyl or heteroaryl-heteroalkyl, a(C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a (C₄-C₁₀) alkyleno orheteroalkyleno.
 20. A method of claim 17 wherein the reaction is carriedout in a single container to yield the oxysterol having the formula

or a pharmaceutically acceptable salt, hydrate or solvate thereof.